Use of aplidine for the treatment of pancreatic cancer

ABSTRACT

Aplidine is active against cancer of the pancreas, including metastatic pancreatic cancer.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy of cancer. Theinvention is more specifically related to the use of aplidine for thetreatment of pancreatic cancer.

BACKGROUND OF THE INVENTION

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention and/or treatmentis currently available.

Current therapies, which are generally based on a combination ofchemotherapy or surgery and radiation, continue to prove inadequate inmany patients.

Located in the upper abdomen in the retroperitoneum, the pancreas isassociated intimately with many major structures including the portalvein, stomach, duodenum, common bile duct and the superior mesentericartery.

Pancreatic cancer is the fifth leading cause of cancer death in theUnited States. It is more common among men, and men between the ages of60 and 70 are most at risk. As the tumour grows, the patient's symptomsresult from tumour infiltration of surrounding structure causing pain,nausea, vomiting, weight loss and jaundice. The latter conditionpresents symptoms in no more than one half of the patients. Once tumourinfiltration occurs other structures such as the portal vein becomeaffected and this precludes curative resectioning of the pancreas.

Effective treatment of pancreas cancer must achieve two difficult goals:control of the primary tumour mass, both initially and subsequently, andtreatment of the metastatic tumour cells. As a result of its insidiousonset, the diagnosis of pancreas cancer is delayed frequently forseveral months. This delay has profound implications, since metastaticspread to the liver or lymph nodes has been observed at a time ofdiagnosis in 60% of patients, and this factor diminishes the prospectfor long-term survival. Also, the carcinoma of the pancreas isasymptomatic in its early stage. The most common symptoms at later stageare weight loss, abdominal pain, and jaundice. Weight loss, the causesof which are not fully understood, usually is significant. Jaundiceoccurs if the cancer blocks the common bile duct. By the time themalignant tumour is identified, it often has spread (metastasized) toother parts of the body. The median survival is little more than sixmonths from the time of diagnosis.

Current therapies for this common and difficult-to-treat disease includesurgery and/or chemotherapy. Often the tumour cannot be removed bysurgery, either because it has invaded vital structures that cannot beremoved or because it has spread to distant sites.

Although 5-year survival rates after surgical removal of the pancreasand a large portion of the duodenum have improved, the procedure is onlyused on 9% of patients. Of these, the highest reported 5-year survivalrate is in the range of 20%.

Patients with advanced pancreatic cancer are treated primarily bychemotherapy. The objective of such therapy is to prolong patientsurvival. Surgery and irradiation are used as well to relieve pain andreduce organ blockage.

In spite of considerable research into therapies for these and othercancers, pancreatic cancer remains difficult to treat effectively.

Accordingly, there is a need in the art for improved methods fortreating primary and metastatic pancreatic cancers. The presentinvention fulfills these needs and further provides other relatedadvantages.

Dehydrodidemnin B, now known as aplidine, is the subject of W091/04985.

Further information on aplidine is to be found in, for example: Jimeno,J., “Exploitation of marine microorganisms and invertebrates: Anticancerdrugs from marine origin”, IBC Conf Discov Drugs from Nat NovelApproaches New Sources (Dec. 8-9, London) 1994, 1994; Faircloth, G. etal., “Dehydrodidemnin B (DDM) a new marine derived anticancer agent(MDA) with activity against experimental tumour models”, 9th NCI-EORTCSymp New Drugs Cancer Ther (Mar. 12-15, Amsterdam) 1996, Abst 111;Sakai, R. et al., “Structure-activity relationships of the didemnins”,Journal of Medicinal Chemistry 1996, 39 (14): 2819; Urdiales, J. L. etal. “Antiproliferative effect of dehydrodidemnin B (DDB), a depsipeptideisolated from Mediterranean tunicates”, Cancer Letters 1996, 102 (1-2):31; Faircloth, G. et al., “Preclinical characterization of aplidine(APD), a new marine anticancer depsipeptide (MADEP)”, Proc Amer AssocCancer Res. 1997, 38: Abst 692; Depenbrock, H. et al. “In vitro activityof aplidine, a new marine-derived anti-cancer compound, on freshlyexplanted clonogenic human tumour cells and haematopoietic precursorcells”, British Journal of Cancer 1998, 78 (6): 739; Faircloth, G. etal., “Aplidine (aplidine) is a novel marine-derived depsipeptide with invivo antitumour activity”, Proc Amer Assoc Cancer Res 1998, 39: Abst1551; Faircloth, G. et al., “Preclinical development of aplidine, anovel marine derived agent with potent antitumour activity”, 10thNCI-EORTC Symp. New Drugs Cancer Ther (Jun. 16-19, Amsterdam) 1998, Abst129; Geldof A A, Mastbergen S C, Henrar R E, Faircloth G T.“Cytotoxicity and neurocytotoxicity of new marine anticancer agentsevaluated using in vitro assays”, Cancer Chemother Pharmacol1999;44(4):312-8; Jimeno J, Smith B, Grant W, Faircloth G T. “Acorrelation of selective antitumour activities of the marine-derivedcompound aplidine using different models” 10^(th) NCI-EORTC-AACRSymposium on molecular targets and cancer therapeutics. Washington,1999. Abstract 311; Broggini M. Marchini S, D'Incalci M, Taraboletti G,Giavazzi R, Faircloth G, Jimeno J. “Aplidine blocks VEGF secretion andVEGF/VEGF-RI autocrine loop in a human leukemic cell line”, 11^(th)NCI-EORTC-AACR Symposium on new drugs in cancer therapy. Amsterdam,2000. Abstract 21; Luber-Narod, J., Jimeno, J.; Faircloth, G T. et al.“In vitro safety profile of aplidine, a marine natural product withchemotherapeutic potential”, Proceedings of the AACR, vol. 42, abstract374, March 2001.

In preclinical studies, aplidine had dose-dependent cytotoxic activityagainst the two epithelial-like cell lines, CT-1 and CT-2, and the humancolon cancer cell line, HT-29. The most proliferative line, CT-2, wasthe most sensitive to aplidine. In addition the compound decreasedornithine decarboxylase activity in all three cell lines (Lobo C, GarciaPozo S G, et al.: “Effect of dehydrodidemnin B on human colon carcinomacell lines”, Anticancer Research. 17: 333-336, January-February 1997).In a similar study, aplidine 50 nmol/L inhibited the growth of thebreast cancer cell lines, MDA-MB231 and MCF-7 by 17 and 47%,respectively.

Continuous exposure to low concentrations of aplidine inhibited thegrowth of a number of tumour cell lines, including non-Hodgkin'slymphoma, melanoma, breast, ovarian and non-small cell lung cancers. Themagnitude of effect was dependent on the time of exposure and appearedto be achievable at non-myelotoxic concentrations. Non-small cell lungcancer, breast cancer and melanoma cell lines were sensitive to acontinuous exposure to aplidine at concentrations of >=0.001 micromol/L.aplidine had similar toxicity to doxorubicin against clonogenichaematopoietic stem cells (Depenbrock H, Peter R, et al.: “In vitroactivity of aplidine, a new marine-derived anti-cancer compound, onfreshly explanted clonogenic human tumour cells and haematopoieticprecursor cells”, British Journal of Cancer. 78: 739-744, No. 6,September 1998).

In general drug therapies are evaluated with respect to treating humancancer, e.g., human cancer xenograft lines. Human tumours are seriallyheterotransplanted into immunodeficient, so-called “nude” mice, and themice then tested for their responsiveness to a specific drug.(Giovanella, B. C., et al., Cancer 52(7):1146 (1983)). The data obtainedin these studies strongly support the validity of heterotransplantedhuman tumours into immunodeficient mammals, such as nude mice, as apredictive model for testing the effectiveness of anticancer agents.

Aplidine had significant activity against mice bearing human cancerxenografts. At a maximum tolerated dose of 2.1 mg/kg, aplidine producednear complete remissions in some animals with a treated/control (T/C)tumour ratio of 9%. At 1.25 mg/kg, significant activity was seen againstgastric tumours (T/C 14%) and prostate tumour growth inhibition was alsoobserved (T/C 25%) (Faircloth G. Grant W, et al. “Preclinicaldevelopment of aplidine, a novel marine derived agent with potentantitumour activity”, Annals of Oncology. 9 (Suppl. 2): 34, 1998).aplidine was also active against P388 leukaemia and B16 melanomaimplanted in mice, with an optimal dose of 160 micro/kg. Unlike didemninB, aplidine was active in SC implanted lewis lung carcinomas (FairclothG, Rinehart K, et al.: “Dehydrodidemnin B a new marine derivedanticancer agent with activity against experimental tumour models”,Annals of Oncology. 7 (Suppl. 1): 34, 1996).

The use of aplidine for the treatment of different cancers is describedin WO 0135974 of the present applicant. Examples 17 and 18 of thisapplication state that aplidine was tested in phase I clinical trials todetermine toxicity (Maximum Tolerated Dose) and pharmacokinetic data.Some of the patients had pancreatic tumour, although no hint of activityfor these tumours was reported. It is the case that these 4 patients didnot respond to the aplidine that was administered to them.

SUMMARY OF THE INVENTION

We have for the first time established that aplidine induces anantitumour effect in primary tumours and metastases in human pancreaticcarcinoma models, in a tumour specific manner.

Thus, the present invention provides a method of treating any mammal, inparticular a human, affected by pancreatic cancer which comprisesadministering to the affected individual a therapeutically effectiveamount of aplidine, or a pharmaceutical composition thereof.

The present invention provides a method of treating any mammal, inparticular a human, affected by metastatic pancreatic cancer whichcomprises administering to the affected individual a therapeuticallyeffective amount of aplidine, or a pharmaceutical composition thereof.

The present invention is also directed to the use of aplidine in themanufacture of a medicament for the treatment of pancreatic cancer, inparticular metastatic pancreatic cancer.

DESCRIPTION OF THE FIGURES

FIG. 1. Aplidine induces necrotic and apoptotic cell death in the NP18(panels A-H) and NP9 (panels I-P) tumours only at the ORT site: H&Estaining of ORT NP18 aplidine-treated tumours showed scar fiberreplacement of necrotic tissue (panel B), which was not observed in theORT control group (panel A), nor in treated (panel D) or control (panelC) SC tumours. Hoescht staining of viable ORT NP18 treated tumoursdepicted nuclear condensation (panel F). No induction of nuclearcondensation was observed in control ORT (panel E), SC treated (panel G)or SC control (panel H) tumours. In the ORT NP9 treated tumour, weobserved multiple foci of necrosis occupying most of the tumour (panelJ). These foci were significantly smaller or not observed in the controlORT (panel I), nor in the treated (panel L) or control (panel K) SCgroups. Nuclear condensation was only observed in the viable ORT NP9treated tumours (panel N), but it was not in control ORT (panel M), SCtreated (panel 0) or SC control (panel P) tumours.

FIG. 2. Aplidine activates apoptotic pathways in the NP18 tumour:Decrease AKT activation, activation of caspases 3, 7 and 8 and PARPcleavage were detected in ORT aplidine-treated (T), as compared tocontrol (C) tumours. At the SC site, aplidine did not alter theregulation of any of these proteins. A similar decrease of Bcl-X_(L)protein was detected in ORT or SC aplidine-treated tumours, as comparedwith their respective vehicle controls. No differences in ERK activationwere observed between treated and control tumours in any site.

FIG. 3. Aplidine downregulates cell cycle regulatory molecules in theNP9 tumour: A decreased expression of cyclin B1 and cyclin D1 wasdetected in ORT aplidine-treated (T) as compared with ORT control (C)tumours. In contrast, no differences in cyclin B1 or cyclin D1 levelswere observed between treated and control SC tumours. No active forms ofcaspases 3, 7 or 8 were detected, despite of detecting their respectiveprocaspases in NP9 control (C) or treated (T) tumours; however,downregulation of FAK was observed only in the ORT-treated tumours. ERKactivation was unaltered by treatment in both tumour site. Expression ofbeta-actin was used to control for equal protein loading.

FIG. 4. H&E staining of aplidine-treated NP9 peritoneal metastasis:aplidine-treated implants (Panel A (40×magnification) andB(200×magnification)) did not contain necrotic areas. No differences innecrosis were observed in aplidine-treated implants, as compared tocontrol implants (panel C (×200) and D (×200)). A high percentage oftumour cells with big and white cytoplasm were detected inaplidine-treated (panel B) as compared with control (panel D) implants.

FIG. 5. Molecular analysis of aplidine-treated NP9 peritonealmetastasis: A decreased in ERKs activation was detected only in treated(T) NP9 peritoneal metastasis. No activation of caspase-3 nor PARPproteolysis were detected in control (C) or treated (T) implants. J,Control apoptotic DNA from Jurkat cells treated with camptothecine.

DETAILED DESCRIPTION OF THE INVENTION

Pancreatic cancer in vivo models were tested with aplidine to findevidence of activity. Surprisingly, we found out that aplidine inducesnecrosis, apoptosis and cell cycle arrest in two primary humanpancreatic carcinomas (NP9 and NP18) xenotransplanted in nude mice andvery specially inhibits the growth of the peritoneal metastases in thetested NP9 tumour.

Therefore aplidine, or a pharmaceutical composition thereof, will beeffective in the treatment of pancreatic cancer.

Aplidine has the following formula:

Examples of pharmaceutical compositions containing aplidine includeliquid (solutions, suspensions or emulsions) with suitable compositionfor intravenous administration, and they may contain the pure compoundor in combination with any carrier or other pharmacologically activecompounds.

Solubilised aplidine shows substantial degradation under heat and lightstress testing conditions, and a lyophilised dosage form was developed,see W099/42 125 incorporated herein by reference. In a currentlypreferred embodiment freeze-drying was performed from a 500 mg/mLsolution of aplidine in 40% (v/v) tert-butanol in Water for Injection(Wfl) containing 25 mg/mL D-mannitol as bulking agent. The prototype,containing 500 mg aplidine and 25 mg D-mannitol as bulking agent pervial was found to be the optimal formulation in terms of solubility,length of lyophilisation cycle and dosage requirements in the clinicalstudies. The optimal reconstitution solution was found to be 15/15/70%(v/v/v) Cremaphor EL/ethanol/Wfl (CEW). Both reconstituted product anddilutions (up to 1:100 v/v) of the reconstituted product with normalsaline appeared to be stable for at least 24 hours after preparation.Shelf-life data, available thus far, show that the formulation is stablefor at least 1 year when stored at 4 C in the dark.

Administration of aplidine or compositions of the present invention isbased on a Dosing Protocol preferably by intravenous infusion. We preferthat infusion times of up to 72 hours are used, more preferably 1 to 24hours, with about 1, about 3 or about 24 hours most preferred. Shortinfusion times which allow treatment to be carried out without anovernight stay in hospital are especially desirable. However, infusionmay be around 24 hours or even longer if required. Infusion may becarried out at suitable intervals with varying patterns, illustrativelyonce a week, twice a week, or more frequently per week, repeated eachweek optionally with gaps of typically one week.

Depending on the type of tumour and the developmental stage of thedisease, the treaments of the invention are useful in preventing therisk of developing tumours, in promoting tumour regression, in stoppingtumour growth and/or in preventing metastasis.

The correct dosage of the compound will vary according to the particularformulation, the mode of application, and the particular situs, host andtumour being treated. Other factors like age, body weight, sex, diet,time of administration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severity of the disease shallbe taken into account. Administration can be carried out continuously orperiodically within the maximum tolerated dose. Further guidance isgiven in WO 0135974 which is incorporated herein by reference in itsentirety.

The compound aplidine and compositions of this invention may be usedwith other drugs to provide a combination therapy. The other drugs mayform part of the same composition, or be provided as a separatecomposition for administration at the same time or a different time. Onepreferred drug to be combined with aplidine is carnitine.

Also preferably, aplidine is administered in conjunction with one ormore chemotherapeutic agents effective against pancreatic cancer such asgemcitabine or 5-FU.

According to the present invention, we demonstrated that aplidineinduces necrosis, apoptosis and cell cycle arrest in two primary humanpancreatic carcinomas (NP9 and NP18) xenotransplanted in nude mice andinhibits the growth of the peritoneal metastases in the tested NP9tumour.

Aplidine has antitumour activity against the NP18 tumour, when implantedat the ORT site, through apoptotic and necrotic induction, followed byscar fiber formation. Apoptosis appears to be mediated by the AKTpathway, since we observed inhibition of AKT activation and caspases 3,7 and 8 activation leading to PARP cleavage in these tumours. Othergrowth regulatory pathways, such as the ERKs remained unchanged.Diminished AKT activation is a common finding in drug-induced apoptosis.

Very significantly, aplidine has antitumour activity against the NP9tumour, when implanted at the ORT site, and its mechanism of actionappear to be a combination of induction of growth arrest and cell death.This is based on the observed microscopic necrosis, and on thedownregulation of cyclins D1 and B1 and Ki67 and high percentage ofnuclear condensation, by Hoescht staining, in the viable tissue in thesetumours. Thus, in spite of not observing a reduction in tumour size,this was likely to occur at a later time, as a consequence of theresorption of these necrotic foci. Therefore, aplidine induces atumour-specific alteration of apoptotic and/or proliferative pathwayswhich could explain its antitumour effect when implanted at the ORTsite.

In addition to the antitumour effect of aplidine against the primarypancreatic carcinomas, we show here that this compound is alsoantimetastatic in the NP9 tumour. This antimetastic effect wasdemonstrated, both, when treatment started before metastaticimplantation and by the growth inhibition of previously establishedmetastatic implants.

The metastatic foci that had 1-2 mm before treatment, disappearedcompletely or had undergone a significant reduction in size and becameonly detectable under the microscope, showing no necrotic areas.Therefore, the most likely mechanism for their reduction in size was,not necrosis, but apoptotic induction.

Interestingly the molecular response of metastases and primary tumour tothe drug differed (ERK activation was decreased in metastases andunchanged in the primary tumour); implying that the response of themetastases could not be anticipated from that observed in the primarytumour. Our observation is very relevant because, to date, no effectivetherapy has been described for the treatment of metastatic pancreaticcarcinoma. Based on these findings a phase II clinical trial on exocrinepancreatic cancer was designed.

EXAMPLES

Preliminary Report

Growth Inhibitory and Antimetastatic Activity of Aplidine in HumanPancreatic Carcinoma Xenografts in Nude Mice

Aplidine is a new cyclic depsipeptide with anticancer activity in vitroand in vivo in different tumour types, but it has not been tested inpancreatic carcinoma. Nevertheless, this pathology has a poor prognosisbecause of difficult early diagnosis, aggressiveness and lack ofeffective systemic therapies. Most patients die of metastases present atdiagnosis. We compared the antitumour effect of aplidine against 2 humanpancreatic carcinomas, NP9 and NP18, using orthotopic (ORP) andsubcutaneous (SC) tumour implantation models. NP9 tumour shows anextensive peritoneal dissemination 3-4 weeks after implantation.METHODS: Groups of ten male Nu/Nu Swiss mice. (4 wk old) were treatedwith vehicle or aplidine. To evaluate primary tumour response, ipaplidine was given, starting 2 weeks (wk) after implantation, at 0.8mg/kg q4d for 4 wk (NP9) or at 0.8 mg/kg q4d for 6 wk, ceasing treatmentthe 4th and 5th wk (NP18). To evaluate the response of NP9 metastasisaplidine regime was 0.8 mg/kg q4d per 3 doses, starting 4 weeks afterimplantation. We measured final tumour weight, macroscopic viability(viable cortex/total tumour weight), microscopic necrosis, apoptoticinduction and signal transduction. RESULTS: aplidine showed antitumoureffect on NP9 and NP18 primary tumours, which was exclusively observedat the microscopic and molecular level on the ORT site, not at the SCsite, in both tumours. This effect was due to apoptotic inductionleading to a reduced number of viable cells in both tumours. Apoptoticinduction on NP18 was associated with PARP cleavage and reduced AKTactivation, only in ORT-treated tumours aplidine induced a decrease incyclins B1 and D1 and FAK expression only in ORT NP9-treated tumours. Inaddition, aplidine showed complete inhibition of NP9 peritonealimplants, whether the treatment started before or after the occurrenceof the metastatic deposits. Absence of necrosis and a decrease in MAPKactivity were observed in treated metastases. CONCLUSIONS: The use of anORT implantation model permitted the observation of the antitumoureffect of aplidine on primary tumours and metastases of human pancreaticcarcinomas aplidine altered apoptotic and proliferative pathways, whichcould explain its antitumour effect. Moreover, ORT and SC implantedtumours showed a different response to this drug.

Detailed Report

We used two human pancreatic carcinomas (NP18 and NP9) perpetuated asorthotopic xenografts in nude mice. NP9 produce an extensive peritonealdissemination 4-5 weeks after implantation (metastatic). We implantedthese tumours in four week-old male Nu/Nu Swiss mice (Charles River),housed in autoclaved cages and provided γ-ray sterilized bedding andfood. They were anesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich)and 10 mg fragments of each tumour from previous passages were implantedorthotopically (ORT, at the pancreas). Xenografted animals were randomlydistributed in control (n=10) and experimental (n=10) groups just beforetreatment. We excluded animals bearing non palpable tumours after ORTimplantation, or with tumour volumes lower than 0.13 cm³ (0.5 cm indiameter). Treatment schedule depended on tumour growth rate. In thefast growing NP9 tumour, two schedules of treatment were used. In one,treatment started two weeks after tumour implantation, before peritonealdissemination had occurred. Each animal in the experimental groupreceived ip administrations of aplidine at a dose of 0.8 mg/Kg q4d perfour weeks. In the other schedule, treatment started four weeks aftertumour implantation, once peritoneal dissemination had occurred, with aregime of aplidine of 0.8 mg/Kg q4d per three doses. The existence ofmacroscopically visible implants was confirmed one day before thetreatment begun, by the sacrifice of one extra animal. In the NP18tumour, treatment started 4 weeks after tumour implantation and theschedule of aplidine was 0.8 mg/Kg q4d ip for 4 weeks, ceasing treatmentfor two weeks, and continuing with 0.8 mg/Kg q4d ip for two additionalweeks. The animals in the corresponding control groups received i.p.administrations of vehicle following the same schedule. Afterimplantation, and during treatment, mice were inspected twice a week andweighted weekly to monitor for compound toxicity.

At the end of treatment, animals were euthanized and disseminationrecorded. Primary tumours were excised and weighted in all groups. Tumoraliquots were frozen down in liquid nitrogen or fixed in formaline. Wecounted the number of peritoneal implants in each animal and tooksamples for histological and molecular analysis. The mean of the totaland solid (central necrosis excluded) tumour weight was compared betweengroups, using the Student t test, the differences being significant atp<0.05.

Histological Analysis and Hoescht Test

For histopathological analysis, formalin-fixed paraffin-embedded tissueswere stained with H&E following the standard protocol and the presenceor absence of microscopic cell death were observed. Nuclear staining ofDNA was performed in 5 □m sections of formalin-fixed paraffin-embeddedtumour tissue. Sections were dewaxed in xylene and dehydrated, washedtwice with PBS, permeabilized with triton X-100 for 10 min at RT andwashed twice with PBS. Then, the slides were incubated with Hoescht(1:5.000 in PBS) for 1 h, washed with sterile water and dried at RT.Finally, the slides were mounted and observed under a flourescencemicroscope, analyzing only areas not involving microscopic necrosis.

Western Blot Analysis

Molecular analyses were performed in whole cell protein extracts oftumours by Western blot, as described (11). The anti-active MAPK Ab(Promega) (1:20,000) detected active Erk-1 and Erk-2 forms. The rabbitanti-cyclin B1 (1:1,000), anti-Bcl-XL (1:7,000-o/n), and anti-FAK(diluted 1:7.000) and the goat anti-p-actin (1:15,000) polyclonal Abswere from Santa Cruz. The rabbit anti-Cyclin D1 was from UpstateBiotech. The rabbit anti-caspase-3, anti-caspase-7 and anti-caspase-8Abs were from Pharmingen, the rabbit anti-active-AKT from New EnglandBiolabs, and the rabbit anti-PARP from Boehringer-Mannheim. Allsecondary antibodies (Jackson ImmunoResearch) were used at a dilution of1:10,000.

Immunohistochemistry Analysis

IHC analysis was performed on paraffin-embeded tumour tissue. Five □msections were dewaxed in xylene and dehydrated. Endogenous peroxidaseactivity was blocked with 0.03% H₂O₂. Antigen was retrieved heating andincubating with 10 mM citric acid monophosphate buffer (pH 6.0).Sections were incubated with anti-Ki67 Ab at RT for 1 h and, then, withsecondary Ab peroxidase conjugated EnVision™ (Dako) for 30 min at RT,followed by DAB+ cromogen (Dako) and counterstaining with hematoxyline.Sample quantitation was done counting positively stained cells in threerandom high power (100×) fields. Differences between groups wereconsidered significant at p<0.05 (Student t test).

Results

We first analyzed the effect of aplidine on NP18 and NP9 humanpancreatic primary tumours after aplidine treatment. We describe themacroscopic and microscopic changes, and the alteration of theexpression and/or activation of apoptotic and cell cycle regulatoryproteins. We also describe the effect of aplidine on the metastaticimplants at the macroscopic, microscopic and molecular level.

Effect of Aplidine on the Primary Tumours

Aplidine altered the macroscopic appearance of the NP9 primary tumour atthe ORT site. Tumor margins were very well defined in the ORT treatedgroup, since they did not invade other organs of the peritoneum, makingits dissection easy. In contrast, tumours in ORT control animals invadedthe spleen and the wall of the peritoneum. After aplidine treatment thesize or macroscopic appearance of the NP18 primary tumour did notsignificantly change, as compared with its control.

Effect on Tumour Weight

Once primary tumours were excised we analyzed the effect of aplidine ontumour weight. At the end of the treatment period, there were nosignificant differences in total or solid tumour weight between theaplidine-treated and the control group in both tumours at any site.Final tumour weights are described in Table 1. Total tumour Solid tumourweight (g) weight (g) NP9 SC Control 2.46 ± 0.6^(a) 1.04 ± 0.33^(e)treated 1.56 ± 0.8^(a) 0.87 ± 0.44^(e) ORT control 2.74 ± 0.85^(b) 2.15± 0.51^(f) treated 3.72 ± 1.09^(b) 3.04 ± 1.04^(f) NP18 SC control 17.7± 5.9^(c) — treated 15.5 ± 5.3^(c) — ORT control 9.96 ± 3.5^(d) —treated 9.34 ± 1.3^(d) —^(a, b, c, d, e, f)No significant differences (Student T test)Histopathological Analysis

In the NP18 model, we observed a significantly higher level of necrosisin the ORT treated than in ORT control tumours. Moreover, scar fibersfilled the space left by extensive areas of dead tumour cells,exclusively in the ORT treated tumours. In the NP9 tumour, the foci ofnon-viable/necrotic cells occupied 75-100% of each tumour. In contrast,necrosis occupied only 0-25% of each tumour in control NP9 ORT tumours.Therefore, aplidine significantly increased the areas of microscopicnecrosis in both the NP9 and NP18 primary tumours.

Hoescht Nuclear Staining

The analysis of the histologically viable tissue, in both NP18 and NP9tumours, after nuclear staining with Hoescht dye, revealed enhanceddensity of picnotic nuclei with condensed and fragmented chromatin inthe ORT treated tumours, which were not observed in the ORT controltumours. Therefore, aplidine significantly increased apoptosis in boththe NP18 and NP9 tumours.

Effect of Aplidine on the NP9 Peritoneal Implants

We tested the possible antimetastatic effect of aplidine on the ORT NP9tumour since it produced early and massive peritoneal dissemination. Inthe ORT control group, we recorded more than 100 implants (1-2 mmdiameter) per animal, four weeks after implantation.

After completion of aplidine treatment, which initiated beforemicroscopically visible dissemination had occurred, no peritonealimplants were observed in any animal. In addition, aplidine alsoinhibited the growth of the metastases when treatment started once theywere macroscopically evident, since animals in the treated group hadonly a few (7-20) and small implants (less than 1 mm diameter), whereas,control animals presented more than 50 implants, reaching 3-4 mm ofdiameter. Therefore, aplidine completely abolishes peritonealdissemination of the ORT implanted NP9 tumour, and it is able to inhibitthe growth of already established metastatic implants.

Pathological and Molecular Analysis of NP9-Treated Metastasis

We histopathologically analyzed the macroscopically visible implants inaplidine-treated and vehicle treated animals. Despite the significantlysmaller size of the metastatic foci in experimental than in controlanimals, aplidine-treated metastases did not show higher cell death thancontrol metastases, at the time of the analysis, since tumour tissue inboth group was microscopically viable. Nuclear staining with Hoeschtshowed a few (3-6) apoptotic nuclei per treated implant, but nosignificant differences were observed between aplidine-treated andcontrol metastases in number of apoptotic nuclei. Molecular analysisrevealed that NP9 aplidine-treated had lower ERK activation than controlmetastases. No activation of Caspase-3 nor PARP cleavage were observedin treated metastasis. Thus, the inhibition of growth in metastasesassociated with a reduction in ERK activation.

1. A method of treating a mammal affected by pancreatic cancer whichcomprises administering to the affected individual a therapeuticallyeffective amount of aplidine, or a pharmaceutical composition thereof.2. The method according to claim 1, wherein the pancreatic cancer ismetastatic pancreatic cancer.
 3. The method according to claim 1,wherein the mammal affected by pancreatic cancer is human.
 4. The methodaccording to claim 1, wherein the pancreatic cancer is refractory toother treatments.
 5. The method according to claim 1, wherein theaplidine is adminstered simultaneously or sequentially in combinationwith another drug.
 6. The method according to claim 5, wherein the otherdrug is a chemotherapeutic agent against cancer.
 7. The use of aplidinein the preparation of a medicament for a method according to claim
 1. 8.The method according to claim 2, wherein the mammal affected bypancreatic cancer is human.